Notch signaling is an evolutionary conserved mechanism used by metazoans to direct cell fate decisions, proliferation and apoptosis at all stages of development, including self-renewing adult tissues. Aberrant Notch signaling is implicated in cancer, especially in the emerging field of cancer stem cells. Mammalian cells contain four Notch receptors and five Delta and Jagged cognate ligands;the overall goal of this proposal is to elucidate the significance of context on individual Notch receptor subtype-mediated signals using non-invasive molecular imaging strategies. We propose to develop enabling technologies that will facilitate high throughput screening for agents and gene products that are capable of modulating the Notch signaling pathway in cancer, inherited diseases and facilitate tissue engineering. A comprehensive mechanistic understanding of how to manipulate individual receptors in a context-dependent manner still eludes investigators. We propose to develop a real time imaging system that will go beyond the currently available reporters in providing real time, quantitative accounting of the activation status of individual Notch receptor subtypes. The reporter system we are developing is based on the luciferase complementation imaging technology developed in the ICMIC Molecular Reporter Core at Washington University School of Medicine and enables visualization of the interactions between a specific Notch intracellular domain (NICD) and the common nuclear cofactor RBPjk. The system is versatile;it can be adaptedfor studying the pathway in different cell types and can be easily modified to monitor interaction with other cancer-relevant partners such as components of the NF-kB pathway. While the imaging technology being developed in this application is not directly applicable to imaging in patients as it requires protein engineering, these methodologies will help develop and evaluate novel therapies for Notch-related diseases.